Parathyroid hormone (PTH) is a vital regulator of serum calcium and phosphate levels and one of the few bone-active agents shown to be anabolic in humans, important for osteoporosis therapy. During the past granting period we have developed a high-resolution model for the binding of PTH to its G protein coupled receptor, PTH1R, employing a combination of photoaffinity labeling experiments (providing direct contact points between ligand and receptor) and NMR-based structural studies. In this renewal application, we propose to characterize the bimolecular interactions of PTH1R with the cytosolic signaling (e.g., G proteins) and non-signaling (e.g., NHERF, GRKs) proteins central for the biological actions of PTH. We aim to characterize the determinant structural features of PTH1R for coupling to the G proteins, Gs, Gq/11, and Gi/o, responsible for the signaling properties associated with PTH. We have initiated NMR-based experiments, providing the structure and topological orientation of the cytoplasmic domains of PTH1R while associated with the G proteins. The molecular scaffolding protein, NHERF (Na+/H+ exchange regulatory factor) plays an important role the pharmacological profile of PTH1R, including signaling and receptor life-time (e.g., internalization and desensitization). Here, employing high-resolution NMR methods we will determine the structural features of the bimolecular interaction of NHERF (NHERF1 and NHERF2) with the C-termini of PTH1R and phospholipase C(beta) and use these results to rationally design potent inhibitors of the receptor-protein association. A molecular inhibitor of NHERF-PTH1R inteaction would be a useful physiological tool for detailing the role of this receptor/protein association and could eventually find use as a therapeutic regulator of PTH activity, affording control of the adenylyl cyclase and PLC signaling pathways associated with PTH1R. A final aim is to develop a soluble mimetic of PTH1R, using a 4-helix bundle as templates for the incorporation of the domains of PTH1 required for ligand binding (extracellular face of the receptor) and signaling properties (cytoplasmic face of the receptor). The soluble PTH1R template will allow for biophysical and structural studies as well as high-throughput screening of compound libraries.